DNA polymerases are enzymes that are useful as reagents for genetic engineering and widely utilized for DNA sequencing, labeling, site-directed mutagenesis and the like. Thermostable DNA polymerases have lately attracted attention due to the development of the polymerase chain reaction (PCR) method. Various DNA polymerases suitable for the PCR method have been developed and put on the market.
Currently known DNA polymerases can be generally classified into four families based on the amino acid sequence similarities. Among them, the family A (pol I-type enzymes) and the family B (α-type enzymes) constitute a large majority. DNA polymerases belonging to each one of the families have generally similar biochemical characteristics. However, detailed comparison has revealed that the respective enzymes have properties different from each other in the substrate specificity, the incorporation of a substrate analog, the strength and velocity of primer extension ability, the mode of DNA synthesis, the accompaniment of an exonuclease activity, the optimal reaction conditions (temperature, pH, etc.), the sensitivity to inhibitors or the like. Thus, a DNA polymerase having the most suitable properties for the experimental procedure has been chosen from the existing ones and utilized.
For example, the DNA polymerase derived from Pyrococcus furiosus (Pfu) (see, for example, Patent Documents 1 to 5) is one of the most thermostable DNA polymerases. It has a 3′>5′ exonuclease activity, which is known as a proofreading activity, and it exhibits relatively high fidelity among thermostable enzymes. However, this enzyme have problems that it requires a long time for amplification if it is used for PCR, and it cannot be used for amplification of a long chain because its extension velocity and processivity are low.
Recently, a polymerase called KOD DNA polymerase which has a higher 3′>5′ exonuclease activity, a higher extension velocity and a higher processivity than the Pfu-derived DNA polymerase is commercially available (see, for example, Patent Documents 6 and 7, Non-patent Document 1). It is possible to carry out PCR with high accuracy in a short time using this enzyme. However, this enzyme has problems that it is relatively difficult to determine the reaction conditions because primers or amplification products are degraded due to the strong 3′>5′ exonuclease activity, and it is not suitable for amplification of a long chain.
Furthermore, two types of enzymes have been developed by improving KOD DNA polymerase. One of them, KOD-Plus-DNA polymerase, enables hot-start PCR without a special procedure by adding two monoclonal antibodies to KOD DNA polymerase to suppress the polymerase activity and the 3′>5′ exonuclease activity at normal temperature. The amplification efficiency and the ability of synthesizing a long-chain DNA are increased by optimizing the reaction buffer composition as compared with KOD DNA polymerase while retaining the high fidelity (e.g., Patent Document 8). However, there is a problem that the extension velocity of this enzyme is considerably lower than that of KOD DNA polymerase and is lowered to a level equivalent to that of the Pfu-derived DNA polymerase.
KOD Dash DNA polymerase is a mixture-type DNA polymerase prepared based on the method of Barnes et al. (see, for example, Patent Document 9, Non-patent Document 2). The amplification efficiency and the extension ability are increased by mixing ROD DNA polymerase and a modified type of ROD DNA polymerase from which the 3′>5 exonuclease activity has been eliminated using genetic engineering techniques at an optimal ratio (see, for example, Patent Document 10). Its extension velocity is high like KOD DNA polymerase. However, there is a problem that the fidelity is remarkably decreased as compared with KOD DNA polymerase alone because the 3′>5′ exonuclease activity is relatively decreased.
Patent Document 1: U.S. Pat. No. 5,489,523
Patent Document 2: U.S. Pat. No. 5,545,552
Patent Document 3: U.S. Pat. No. 5,866,395
Patent Document 4: U.S. Pat. No. 6,489,150
Patent Document 5: U.S. Pat. No. 5,948,663
Patent Document 6: U.S. Pat. No. 6,054,301
Patent Document 7: U.S. Pat. No. 6,225,065
Patent Document 8: United States Patent Publication No. 2002/0076768
Patent Document 9: U.S. Pat. No. 5,436,149
Patent Document 10: U.S. Pat. No. 6,008,025
Non-patent Document 1: Barnes W. M., Proc. Natl. Acad. Sci. USA, vol. 91, No. 6, p. 2216-2220 (1994)
Non-patent Document 2: Takagi M., et al., Appl. Environ. Microbiol., vol. 63, No. 11, p. 4504-4510 (1997)